In 2004, Phytophthora symptoms were observed on two different fir
species used for Christmas trees in Norway. Isolations resulted in a
Phytophthora sp. related to P. inundata from relatively newly
established Nordmann fir (Abies nordmanniana) and P. megasperma
from seven-year-old subalpine fir (A. lasiocarpa). The Nordmann
fir plantation was more severely damaged, with approximately 70% of the trees
dead or dying. In the field with subalpine fir, approximately 25% of the trees
had yellow or brown foliage and stem canker. Pathogenicity was proven for both
Phytophthora isolates on seedlings from their respective host plants. The
massive infestation in the Nordmann fir plantation approximately one year after
planting suggests that the pathogen was introduced into the planting with the
transplants. Except for a recent report of P. cambivora on noble fir (A.
procera) produced for bough production (17), Phytophthora has never
been reported before on fir in Norway.

Introduction

Traditionally, Norway spruce (Picea abies) has been the dominant
Christmas tree in Norway; however, firs (Abies spp.) have taken increased
market share over the last two decades. Approximately 1 million Christmas trees
are planted yearly, of which 70% consists of different fir species. The main fir
species is Nordmann fir (A. nordmanniana), but also subalpine fir (A.
lasiocarpa) is common in areas where the climate is too harsh for Nordmann
fir.

Phytophthora citricola and P. citrophthora are known to cause root
rot on Lawson Falsecypress, or Port Orford cedar (Chamaecyparis lawsoniana),
in bough plantations, private gardens, and public parks in Norway (14), but
damage by Phytophthora spp. have not been reported until recently in fir
plantations. P. cambivora was found on noble fir in a bough plantation in
2004 (17), but Phytophthora spp. have never been isolated from forest
stands in Norway.

We have not been able to find any specific reports of Phytophthora
diseases on Christmas trees in Europe. However, significant mortality due to
Phytophthora spp. has occurred in Europe in a wide range of trees and woody
ornamentals belonging to different genera (Aesculus, Tilia, Prunus, Taxus,
Chamaecyparis, Abies, Rhododendron, and Erica), and the species
causing disease have usually been P. cambivora or P. cinnamomi
(2). In the USA numerous Phytophthora spp. on fir species have been
reported. Although variation in ability to cause disease occurs between
different Phytophthora spp., several of the pathogens are associated with
diseased fir species in the USA: P. cactorum, P. cambivora,
P. cinnamomi, P. citricola, P. cryptogea, P. drechsleri, P. gonapodyides, P.
megasperma, and P. pseudotsugae (5,6,7,8,11).

The objective of this investigation was to identify the disease causing
organisms and thereby document the presence of Phytophthora on fir used
for Christmas trees in Norway. Preliminary results from these investigations are
published in proceedings from international meetings (15,16).

Nordmann Fir

Samples were collected in May 2004 in a Nordmann fir Christmas tree field
established in April 2003 (Fig. 1) in Rogaland Co., on the southwestern coast
of Norway. Approximately 70% of the Nordmann fir trees in the field were
symptomatic. Symptoms included poorly developed roots and brownish to red
discoloration under the bark from the stem base downwards (Fig. 2). The foliage
had different stages of drought symptoms: pale green, yellow, or brown.

Fig. 2. Root rot and discoloured stem on Nordmann fir (Abies nordmanniana). The disease was caused by a Phytophthora sp. with ITS rDNA sequences most similar to P. inundata. Photo by V. Talgø.

Isolations were carried out from the area between healthy and diseased
tissue, both from roots and stem bases. The samples were rinsed in running tap
water for approximately 20 min and plated on the Phytophthora selective
medium PARP, both with and without hymexazol. PARP medium contained 17 g
of cornmeal agar, 10 mg of pimaricin, 250 mg of ampicillin, 10 mg of rifampicin, and 100 mg
of PCNB in 1 liter of water. PARPH was prepared by adding 50 mg of hymexazol. A
Phytophthora sp. was isolated on both PARP and PARPH. Isolates
obtained were transferred to V8 agar (1320 ml of distilled water, 330 ml of V-8 juice,
3.3 g of CaCO3, and 24.8 g of agar) (Fig. 3) and examined periodically for
oogonia formation. Agar pieces containing mycelium were placed in Petri dishes
containing autoclaved pond water. The dishes were incubated at 15°C or room
temperature and examined periodically with dissecting microscope for sporangia
formation. The isolate produced nonpapillate sporangia with internal
proliferation. The sexual stage was observed in water, but not on agar. The
oogonia were large (average diameter 49 μm). The oospores were aplerotic and
the antheridia predominantly amphigynous. The isolate grew well at 35°C.

ITS amplification and sequencing were carried out on the isolate. DNA was
extracted using the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA).
Amplification of the ITS (internal transcribed spacer) region of nuclear rDNA
was performed with the primers ITS1 and ITS4 (18) with an initial denaturation
at 94°C for 2 min followed by 30 cycles of 30 s at 94°C, 1 min at 55°C
and 1 min at 72°C. The PCR products were purified with QIA Quick PCR
Purification Kit (QIAGEN Inc., Valencia, CA, USA) and sequenced by the ABI
PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied
Biosystems, Drive Foster City, CA, USA). Both strands of the entire ITS region
were sequenced using the primers ITS1, ITS2, ITS3, and ITS4 (18) and the ABI
PRISM 310 Genetic Analyzer (Applied Biosystems, Drive Foster City, CA, USA).
DNA sequences were analysed with the SeqManä II 4.03 (Lasergene Sequence
Analysis Software, DNASTAR Inc., Madison, WI, USA). The program BLASTN
2.2.1 (1) was used to search existing DNA databases for sequences showing
homology to the bedstraw obtained sequences. Compared to the GenBank the
obtained sequences (accession number EF027352 in GenBank) were most
similar to P. inundata (only 14 base pairs different).

A pathogenesis test to fulfil Koch’s postulates was carried out in autumn
2005. Four Nordmann fir plants (2 to 3 year old) were inoculated with the obtained
isolate. Inoculation was performed by placing agar (V8) containing the organism
in the growth medium close to the roots of the Nordmann fir seedlings. Inoculum
per plant or container equaled the amount grown in one 9 cm Petri dish during six
days. Before inoculation the culture was kept at room temperature and no
artificial light. The stem base on each plant was gently wounded with a scalpel
(removing approximately 0.5 cm² of the outer bark) before inoculation. All
plants were potted in 14-cm containers with limed and fertilized peat as growth
medium. Three plants were kept as controls (wounded, but not inoculated). The
containers were placed in a research greenhouse throughout the experimental
period. No artificial light or heating was used in the greenhouse, and the
temperature fluctuated between 7 and 25°C with outdoor temperatures. The
plants were regularly watered. Requirements for water varied with temperature
and disease development.

Seven weeks after inoculation, all plants were examined for visible symptoms
of stem canker or root rot. All the inoculated plants had reduced root growth
compared to the control, and stem cankers had started to develop; however, none
of the plants had died. Reisolations were carried out from all plants. Diseased
roots were washed in running tap water, and the stem and root pieces were
surface sterilised in 1% NaOCl for 30 s and rinsed in sterile distilled
water before small pieces of diseased tissue were transferred to PARPH medium. A
Phytophthora sp. was isolated from the inoculated plants. The control
plants had no signs of canker, and no Phytophthora growth was obtained
from isolations carried out from the roots of the control plants. Following the
identification of Phytophthora growth, colonies were sub-cultured onto V8
agar before identification to species level. The ITS rDNA sequences of the
reisolated culture was identical to the original culture described above.

The field where the diseased Nordmann fir trees grew had fairly heavy soil.
Drainpipes were installed, so the field was not particularly wet, but average
annual precipitation is high where the field was located (> 2000 mm). Combined
with cool summers and mild winters, this provides very favourable conditions for
Phytophthora spp. The massive infestation in a relatively newly
established field, and the fact that the field had previously only been used for
grass production over decades, strongly indicated that the disease had followed
the imported transplants.

In the USA, P. inundata has been found on fir (4) and has also been
isolated from a number of other host plants in different countries: roots of
horse chestnut (Aesculus hippocastanum) and Salix matsudana in UK,
from river water in France, from alder (Alnus sp.) debris in a pond in
Denmark, from roots of Vitis sp. in South America (3), and from
olive roots (Olea sp.) in Spain (13). Interestingly, the bare root
Nordmann fir transplants used in this plantation were imported from Denmark
where they had been produced in seedling beds in the field. Since P. inundata
was isolated from alder debris in a pond in Denmark, arrangements are currently
being made for sampling soil and plants in the nursery where the seedlings were
produced.

Subalpine Fir

During the summer 2004, several seven-year-old subalpine fir in a plantation
in the southeastern part of Norway (Buskerud Co.) had typical Phytophthora
symptoms and were dying. Diseased trees had distinct, uniform, pale yellow
foliage, and the stem base was girdled (Fig. 4). Many trees had been dead for
some time, and the foliage was completely brown (Fig. 5). None of the trees
showed any flagging symptoms (dead basal branches on cankered parts of the
stem).

Isolation and identification methods were identical to those carried out for
Nordmann fir, except that the Petri dishes with the isolated culture in
autoclaved pond water were kept at room temperature. The water was changed twice
to stimulate sporangia formation. When the first sporangia were observed, the
dishes were placed at 8°C and darkness for 4 h to stimulate sporulation.
Two isolates were examined from subalpine fir. Both produced non-caduceous and
non- papillate sporangia with internal proliferation. One isolate did not
produce oogonia in agar. The other isolate readily produced oogonia on agar
(both on PARPH and V8). The oogonia producing culture had less fluffy/aerial
mycelium than the culture that did not produce oogonia (Fig. 6). The average
diameter of the oogonia was 48 μm on V8. The oospores were aplerotic with an
average diameter of 40 μm. The antheridia were paragynous, but a few amphigynous
antheridia were also observed. All morphological characters were compared to
descriptions made by Erwin and Ribeiro (9). The sequencing of isolates took
place as described for Nordmann fir, and both isolates (accession number
EF027353 in GenBank) were confirmed to be 100% identical to P. megasperma
by ITS rDNA sequencing.

Fig. 6. Two different isolates of Phytophthora megasperma from subalpine fir (Abies lasiocarpa) on V8 agar. The culture to the left has fluffy appearing mycelium and does not produce oogonia on agar. The culture to the right readily produces oogonia on agar and has very little aerial mycelium. Photo by V. Talgø.

Inoculation tests were carried out during winter 2006 with the two
cultures obtained from the diseased subalpine fir on subalpine seedlings in the
same way as described for Nordmann fir, except that only three seedlings were
used per isolate. The plants were kept at 18°C in natural light. Reisolations
were carried out after seven weeks from one of the inoculated plants per
isolate. The remaining two inoculated plants per isolate were kept longer to
observe symptom development. Both isolates gave identical symptoms: reduced root
growth and stem canker. Reisolated cultures were identical to the originals.
Oogonia were only produced on culture reisolated from the plant that had been
inoculated with the oogonia producing culture.

The Phytophthora-infected subalpine fir trees were planted on sloping
terrain, and approximately 25% of the trees were dead or dying in a section in
the middle of the field, indicating that inoculum may have moved downhill by
drainage or surface water. Since the trees already had been in the field for
seven years, it was not possible to verify whether the disease was brought in by
the transplants or not.

Flat areas of the farm had been used for intensive vegetable production, and
there had been problems with Phytophthora on Chinese cabbage (Brassica
rapa var. pekinensis) some years ago, but the Phytophthora had
not been identified to species. P. megasperma and other Phytophthora
spp. are reported on Brassica sp. (12), and thus a possible dissemination
of Phytophthora from the vegetable field to the adjacent subalpine fir
plantation may have occurred.

P. megasperma is associated with diseased roots and stems of Frasier fir
(A. fraseri) in the USA (11), but greenhouse pathogenicity assays in
Michigan indicated that this pathogen is not very aggressive (10). The
inoculated subalpine fir plants reported here had started to develop stem
canker, and the root growth was reduced. However, compared to noble fir plants
eleven weeks after they were inoculated with P. cambivora (17), the symptoms were less severe on the subalpine fir plants inoculated with
P. megasperma and kept for the same length of time.

Current Situation

After the findings of Phytophthora spp. on fir in 2004, we have
provided information about Phytophthora-symptoms to the growers and the
extension service in grower journals in Norway and Denmark and meetings for
Christmas tree growers, but we have not received any new reports about
Phytophthora-symptoms on fir in Norway. Therefore, we believe the pathogens
are not widespread on fir in Christmas tree or bough plantations in the country.

Efforts are undertaken to reduce the risk of introducing Phytophthora
spp. to new fields from the infected fields or by infected nursery stock. The
majority of the seedlings in Norwegian forest nurseries are grown in containers
placed on benches above ground; thus, depending on other sanitary measures in
the nurseries, they are protected to a certain extent against soilborne damaging
agents such as oomycetes, fungi, and nematodes. Importation of bare root plants
is considered to be the greatest threat, and growers are advised to thoroughly
inspect transplants prior to planting.

Acknowledgments

We want to thank Terje Pundsnes in the Christmas tree extension service
(Norsk Pyntegrønt Forsøksring) for sampling plant material. We also want to
thank Trude Slørstad, Grete Lund, and Eliza B. Gauslaa at Norwegian Institute for
Agricultural and Environmental Research for their valuable technical assistance.